Transmembrane and truncated (SEC) isoforms of MUC1 in the human endometrium and Fallopian tube

The cell surface mucin MUC1 is expressed by endometrial epithelial cells with increased abundance in the secretory phase of the menstrual cycle, when it is found both at the apical cell surface and in secretions. This suggests the presence of a maternal cell surface glycoprotein barrier to embryo implantation, arising from the anti-adhesive property of MUC1. In previous work, we demonstrated alternatively spliced MUC1 variant forms in tumour cells. The variant MUC1/SEC lacks the transmembrane and cytoplasmic sequences found in the full-length variant. We now show that MUC1/SEC mRNA is present in endometrial carcinoma cell lines, endometrial tissue and primary cultured endometrial epithelial cells. The protein can be detected using isoform-specific antibodies in uterine flushings, suggesting release from endometrium in vivo. However, on the basis of immunolocalisation studies, MUC1/SEC also remains associated with the apical epithelial surface both in tissue and in cultured cells. Transmembrane MUC1 and MUC1/SEC are both strikingly localised to the apical surface of tubal epithelium. Thus MUC1 may contribute to the anti-adhesive character of the tubal surface, inhibiting ectopic implantation. The mechanism by which this barrier is overcome in endometrium at implantation is the subject of ongoing investigation.     

MUC16 is produced in tracheal surface epithelium and submucosal glands and is present in secretions from normal human airway and cultured bronchial epithelial cells

The gel-forming MUC5AC and MUC5B mucins have been identified as major components of human airway mucus but it is not known whether additional mucin species, possibly with other functions, are also present. MUC16 mucin is a well-known serum marker for ovarian cancer, but the molecule has also been found on the ocular surface and in cervical secretions suggesting that it may play a role on the normal mucosal surface. In this investigation, the LUM16-2 antiserum (raised against a sequence in the N-terminal repeat domain) recognized MUC16 in goblet and submucosal gland mucous cells as well as on the epithelial surface of human tracheal tissue suggesting that the mucin originates from secretory cells. MUC16 mucin was present in 'normal' respiratory tract mucus as well as in secretions from normal human bronchial epithelial (NHBE) cells. MUC16 from NHBE cells was a high-molecular-mass, monomeric mucin which gave rise to large glycopeptides after proteolysis. N- and C-terminal fragments of the molecule were separated on gel electrophoresis showing that the MUC16 apoprotein undergoes a cleavage between these domains, possibly in the SEA domain as demonstrated for other transmembrane mucins; MUC1 and MUC3. After metabolic labeling of NHBE cells, most of the secreted monomeric, high-molecular-mass [(35)S]sulphate-labelled molecules were immunoprecipitated with the OC125 antibody indicating that MUC16 is the major [(35)S]sulphate-labelled mucin in NHBE cell secretions.     

Identification of a Moraxella catarrhalis gene that confers adherence to various human epithelial cell lines in vitro

Moraxella catarrhalis is a pathogen of the human airways. We found that expression of the M. catarrhalis gene mcmA by Escherichia coli increases adherence to epithelial cells 100-fold. Furthermore, we discovered that disrupting mcmA decreases M. catarrhalis adherence to laryngeal and lung cells, which are relevant to pathogenesis by the bacterium.     

The UL16 gene of human cytomegalovirus encodes a glycoprotein that is dispensable for growth in vitro.

The UL16 gene of human cytomegalovirus (HCMV) encodes a predicted translation product with features characteristic of glycoproteins (signal and anchor sequences and eight potential N-linked glycosylation sites). Antisera were raised against the UL16 gene product expressed in Escherichia coli as a beta-galactosidase fusion protein. The antisera detected a 50-kDa glycoprotein in HCMV-infected cells that was absent from purified virions. The UL16 glycoprotein was synthesized at early times after infection and accumulated to the highest levels at late times after infection. A recombinant HCMV in which UL16 coding sequences were interrupted by a lacZ expression cassette was constructed by insertional mutagenesis. Analysis of the phenotype of the recombinant virus indicated that the UL16 gene product is nonessential for virus infectivity and growth in tissue culture.     

Direct evidence from siRNA-directed "knock down" that p16(INK4a) is required for human fibroblast senescence and for limiting ras-induced epithelial cell proliferation

The selective pressure for disruption of the cyclin-dependent kinase inhibitor p16(INK4a) in human cancer has been postulated to reflect its role in mediating growth arrest, both in response to telomere erosion (replicative senescence) and to oncogene-induced and other "stress" signals. Given the known species-specific differences in regulation of senescence, we have tested this hypothesis in human, as opposed to rodent, cells by designing a small interfering RNA (siRNA) to knock down p16(INK4a) expression. Transfection of this siRNA into late-passage normal human diploid fibroblasts allowed at least temporary escape from entry into replicative senescence. Furthermore, in our in vitro model of early-stage, RAS-induced thyroid tumorigenesis, sequential transfections with this siRNA allowed outgrowth of small clusters of proliferating epithelial cells, consistent with escape from the spontaneous "senescence", which normally curtails their proliferative response to mutant RAS. These data provide the first direct evidence that p16(INK4a) is necessary for the initiation of both telomere-dependent and telomere-independent senescence in human cells.     

New molecular evidence that Teladorsagia circumcincta (Nematoda: Trichostrongylidea) is a species complex

In the polymorphic Teladorsagia circumcincta (morphs circumcincta and trifurcata), a sheep and goat line (SGL) and a goat line (GL) have been previously described on the basis of the malate dehydrogenase allozyme polymorphism (MDH-2) and of the morphology of the dorsal ray. The GL were never found alone in 1 host, so the status of species was not given to these 2 lines. To investigate further whether there are other genetic markers that will delineate them, we collected T. circumcincta worms from goat and sheep at 8 farms in Touraine (west-central France). The worms were identified individually as being SGL or GL on the basis of MDH-2 polymorphism. This distinctiveness was corroborated by sequences of the beta-tubulin isotype I gene, the second internal transcribed spacer (ITS2) of their rDNA, and the nicotinamide dehydrogenase (ND4) gene of their mDNA. The extent of the divergence in the 3 additional genetic markers was such that SGL and GL may be considered as 2 species. A third putative species was found in the SGL line based exclusively on the ND4 gene. These findings suggest that T. circumcincta is a species complex and that further investigation is required on a wider geographic scale.     

Ovulation in the perfused ovary in vitro: further evidence that estrogen is not required

The effect of inhibition of estrogen synthesis on ovulation in rat ovaries perfused in vitro with medium without phenol red was examined. The addition of luteinizing hormone (LH, 0.1 microgram/mL) plus 3-isobutyl-1-methylxanthine (IBMX, 0.2 mM) to phenol red-free perfusion medium (M199 + 4% bovine serum albumin) induced ovulation. The number of ovulations was similar to that found in medium containing phenol red. There was a similar increase in estradiol (1, 3, 5 (10)-estratriene-3, 17 beta-diol) levels in the medium in both groups. The addition of 4-hydroxy-4-androstene-3, 17-dione (4-OH-A, 5 microM) to phenol red-free medium blocked the increase in estradiol levels induced by LH + IBMX, but did not prevent ovulation. There was no significant difference in the number of ovulations in the three groups. In conclusion, phenol red in the perfusion medium does not influence ovulation induced by LH + IBMX. Furthermore, an increase in estrogen is not required during the immediate preovulatory period for ovulation to occur.     

Placentation in the degu (Octodon degus): analogies with extrasubplacental trophoblast and human extravillous trophoblast

This study examined the placentation in the degu, the origin of the extrasubplacental trophoblast (EST) (extravillous trophoblast in human), and the activity of Na+/K+ ATPase in the placental barrier during different gestational ages, as part of a wider effort to understand the reproductive biology of this species. Fifteen degus at the first stage of gestation, midgestation and at term of pregnancy were studied. At day 27 of gestation, the subplacenta is formed under the wall of the central excavation. Simultaneously, the outermost trophoblast of the ectoplacental cone differentiated into secondary trophoblast giant cells that lie on the outside of the placenta, forming an interface with the maternal cells in the decidua. These giant cells immunostained positive for cytokeratin (CK) and placental lactogen (hPL) until term. During this period, the EST merged from the subplacenta to the decidua and immunostained negative for CK, but at term, immunostained for CK and hPL in the maternal vessels. The vascular mesenchyme of the central excavation invaded the chorioallantoic placenta during this period, forming two fetal lobules of labyrinthine-fine syncytium, the zone of the placental barrier. The activity of Na+/K+ ATPase in the placental barrier was constant during the gestational period. The residual syncytium at the periphery of the placental disc and between the lobules was not invaded by fetal mesenchyme and formed the marginal and interlobular labyrinthine syncytium that immunostained first for CK, and later for hPL, as in the labyrinthine fine syncytium. The presence of intracytoplasmic electron-dense material in the interlobular labyrinthine syncytium suggested a secretory process in these cells that are bathed in maternal blood. Placentas obtained from vaginal births presented a large, single lobe, absence of the subplacenta, and a reduced interlobular labyrinthine syncytium. At day 27, the inverted visceral yolk sac is observed and its columnar epithelium immunostained for CK and hPL. This suggests that the yolk sac is an early secretory organ. The epithelium of the parietal yolk sac covers the placenta. The origin of the EST in the degu placenta and its migration to maternal vessels allows us to present this animal model for the study of pregnancy pathologies related to alterations in the migration of the extravillous trophoblast.     

V(D)J recombination: evidence that a replicative mechanism is not required.

We examined a series of extrachromosomal DNA substrates for V(D)J recombination under replicating and nonreplicating conditions. Complete and partial replications were examined by monitoring the loss of prokaryote-specific adenine methylation at 14 to 22 MboI-DpnI restriction sites (GATC) on the substrates. Some of these sites are within 2 bases of the signal sequence ends. We found that neither coding joint nor signal joint formation requires substrate replication. After ruling out replication as a substrate requirement, we determined whether replication had any effect on the efficiency of V(D)J recombination. Quantitation of V(D)J recombination efficiency on nonreplicating substrates requires some method of monitoring the entry of substrate molecules into the cells. We devised such a method by monitoring DNA repair of substrates into which we had substituted deoxyuridine for 10 to 20% of the thymidine nucleotides in the DNA. The substrates which enter the lymphoid cells were repaired efficiently in vivo by the eukaryotic uracil DNA repair system. Upon plasmid harvest, we distinguished repaired (entered) from unrepaired (not entered) plasmids by cleaving unrepaired molecules with uracil DNA glycoylase and Escherichia coli endonuclease IV in vitro. This method of monitoring DNA entry does not appear to underestimate or overestimate the amount of DNA entry. By using this method, we found no significant quantitative effect of DNA replication on V(D)J recombination efficiency.     

Immunoregulatory properties of fractions from human pregnancy urine: evidence that human chorionic gonadotropin is not responsible.

The immunologically privileged position of the histoin-compatible fetus and placenta is a striking example of a physiologic immunoregulatory mechanism. This study was designed to examine the effects of human chorionic gonadotropin (HCG) on the recognitive proliferative phase and the cytotoxic effector phase of in vitro cell-mediated immune responsiveness, since HCG has previously been reported to be immunosuppressive in vitro and in vivo. Commercial preparations of HCG were found to be potent inhibitors of lymphocyte proliferative responses to nonspecific mitogens like phytohemagglutinin (PHA), specific antigens such as streptolysin-O (SLO), and allogeneic cells as measured in the one-way mixed leukocyte response. Cytotoxic effector function of lymphocytes as measured by antibody-dependent cellular cytotoxicity (ADCC) and mitogen-induced cellular cytotoxicity were also markedly inhibited by these preparations. However, the 50% inhibitory concentration varied widely from lot to lot of these commercial materials. After dialysis, a portion of the inhibitory activity was lost from some but not all HCG lots. The dialysate from those lots with diminished activity was found to be immunosuppressive in vitro but contained no HCG detectable by radioimmunoassay. Following dialysis, the immunosuppressive activity of the various HCG lots remained variable and correlated poorly with values for HCG obtained by a double antibody radioimmunoassay. HCG preparations purified to a homogeneity sufficient for amino acid sequence were found to be only minimally immunosuppressive to the in vitro PHA response and had almost no effect on proliferative responses to antigens and allogeneic cells. These data do not support the concept of a primary immunoregulatory role for HCG, but they suggest that other uncharacterized compounds partially co-purified from pregnant urine along with HCG may have such immunoregulatory activity. Further characterization and identification of this immunoregulatory material(s) is essential, since it appears to have many of the properties of an ideal immunosuppressive compound: a) nontoxicity, b) ready reversibility, c) activity at very low concentration, and d) activity on a broad range of cellular immune functions.     

Glycoconjugates of the human sperm surface: Distribution and alterations that accompany capacitation in vitro

We have studied changes in the binding of fluoresceinated lectins to human sperm during in vitro capacitation. We first determined the surface labeling pattern of viable sperm obtained by the swim-up procedure. Sperm were labeled with 100 μg/ml FITC-conjugated lectin at 4°C for 30 min. We simultaneously used Hoechst stain 33258 as a supravital stain to help differentiate surface from intracellular lectin labeling. Of 14 lectins studied, six (phytohemagglutinin-E, concanavalin A, Ricinus communis agglutinin-I, and the lectins of wheat germ, Lens culinaris, and Pisum sativum) bound to the entire surface of sperm, sometimes with minor local heterogeneity. Three lectins (from peanut, Maclura pomifera, and soybean) usually bound in a punctate manner, with more label on the tail than on the head. Five lectins (Ulex europaeus, Dolichos biflorus, Helix pomatia, and Vicia villosa lectins, and lectin II of Griffonia simplicifolia) bound very poorly or not at all to the sperm surface. Sperm were also inspected for changes in surface lectin binding patterns after 0, 5, and 23 hr of incubation in a capacitating medium. Two lectins showed reproducible changes. The labeling by Maclura pomifera agglutinin decreased by 5 hr in eight of ten experiments, and among sperm labeled with concanavalin A, the incidence of sperm with a highly fluorescent anterior margin of the sperm head increased by about 3.5-fold between 0 and 5 hr. The labeling pattern of the other lectins did not change.     

Direct evidence that all three histidine residues coordinate to Cu(II) in amyloid-beta1-16

We provide direct evidence that all three histidine residues in amyloid-beta 1-16 (Abeta 1-16) coordinate to Cu(II). In our approach, we generate Abeta 1-16 analogues, in each of which a selected histidine residue is isotopically enriched with (15)N. Pulsed electron spin resonance (ESR) experiments such as electron spin echo envelope modulation (ESEEM) and hyperfine sublevel correlation (HYSCORE) spectroscopy clearly show that all three histidine imidazole rings at positions 6, 13 and 14 in Abeta 1-16 bind to Cu(II). The method employed here does not require either chemical side chain modification or amino acid residue replacement, each of which is traditionally used to determine whether an amino acid residue in a protein binds to a metal ion. We find that the histidine coordination in the Abeta 1-16 peptide is independent of the Cu(II)-to-peptide ratio, which is in contrast to the Abeta 1-40 peptide. The ESR results also suggest tight binding between the histidine residues and the Cu(II) ion, which is likely the reason for the high binding affinity of the Abeta peptide for Cu(II).     

A CD8 polypeptide that is lost after passing the Golgi but before reaching the cell surface: a novel sorting mechanism.

The murine CD8 T cell differentiation antigen is a glycoprotein expressed on the cell surface as a heterodimer comprising the products of two closely linked genes, Ly-2 and Ly-3. The Ly-2 gene encodes, through a mechanism of alternate splicing, two polypeptide chains, alpha and alpha', that differ from one another in the lengths of their cytoplasmic tails. All T cells transcribe and translate both the alpha and alpha' polypeptides of Ly-2 and form heterodimers of each of these polypeptides disulphide-bonded with the Ly-3 polypeptide. However, there is very specific, developmentally controlled regulation of the expression of these heterodimers on the cell surface. Namely, immature T cells show no discrimination of CD8 molecules and express on their cell surface heterodimers containing the Ly-3 polypeptide linked to either the alpha or alpha' chain of Ly-2. In contrast, mature T cells express on their cell surface predominantly the heterodimer containing the Ly-2 alpha chain, the species which has a cytoplasmic tail. Moreover, in mature T cells the complexes which contain the alpha' chain are retained within the cell late in processing. These data emphasize the importance of the CD8 accessory molecule in the development of the functional T cell repertoire and uncover a novel protein sorting mechanism in mature T cells.     

Molecular evidence that the proliferative kidney disease organism unknown (PKX) is a myxosporean.

The proliferative kidney organism unknown (PKX), a serious salmonid fish pathogen, is considered to be a myxosporean on the basis of ultrastructural studies, but its real taxonomic position has never been confirmed. In order to ascertain its position, genomic DNA was extracted from PKX and small subunit (SSU) ribosomal DNA was amplified by PCR, cloned and sequenced. A phylogenetical analysis on SSU rDNA from 76 or 128 eucaryotic species was carried out. Whatever the tree reconstruction methods used, PKX was found to be a sister group of the Myxozoa phylum, providing the first molecular evidence for its membership in this phylum.     

Placentation in the degu (Octodon degus): Analogies with extrasubplacental trophoblast and human extravillous trophoblast

This study examined the placentation in the degu, the origin of the extrasubplacental trophoblast (EST) (extravillous trophoblast in human), and the activity of Na⁺/K⁺ ATPase in the placental barrier during different gestational ages, as part of a wider effort to understand the reproductive biology of this species. Fifteen degus at the first stage of gestation, midgestation and at term of pregnancy were studied. At day 27 of gestation, the subplacenta is formed under the wall of the central excavation. Simultaneously, the outermost trophoblast of the ectoplacental cone differentiated into secondary trophoblast giant cells that lie on the outside of the placenta, forming an interface with the maternal cells in the decidua. These giant cells immunostained positive for cytokeratin (CK) and placental lactogen (hPL) until term. During this period, the EST merged from the subplacenta to the decidua and immunostained negative for CK, but at term, immunostained for CK and hPL in the maternal vessels. The vascular mesenchyme of the central excavation invaded the chorioallantoic placenta during this period, forming two fetal lobules of labyrinthine-fine syncytium, the zone of the placental barrier. The activity of Na⁺/K⁺ ATPase in the placental barrier was constant during the gestational period. The residual syncytium at the periphery of the placental disc and between the lobules was not invaded by fetal mesenchyme and formed the marginal and interlobular labyrinthine syncytium that immunostained first for CK, and later for hPL, as in the labyrinthine fine syncytium. The presence of intracytoplasmic electron-dense material in the interlobular labyrinthine syncytium suggested a secretory process in these cells that are bathed in maternal blood. Placentas obtained from vaginal births presented a large, single lobe, absence of the subplacenta, and a reduced interlobular labyrinthine syncytium. At day 27, the inverted visceral yolk sac is observed and its columnar epithelium immunostained for CK and hPL. This suggests that the yolk sac is an early secretory organ. The epithelium of the parietal yolk sac covers the placenta. The origin of the EST in the degu placenta and its migration to maternal vessels allows us to present this animal model for the study of pregnancy pathologies related to alterations in the migration of the extravillous trophoblast.     

Structural evidence that scytalidolisin (formerly scytalidopepsin A) is a serine-carboxyl peptidase of the sedolisin family

Scytalidopepsin A, a pepstatin-insensitive acid endopeptidase from the fungus Scytalidium lignicolum was found to be a member of the sedolisin family of serine-carboxyl peptidases through analyses of the amino acid sequences of peptides derived from the reduced, pyridylethylated enzyme by enzymatic digestion. Hence it should be renamed scytalidolisin (or Scytalidium sedolisin).     

Organ culture of osteopetrotic (ia) rat bone: evidence that the defect is cellular.

Calvarial bone from osteopetrotic (ia) rats and normal littermates has been cultured in a chemically defined medium supplemented with homologous serum to test for the presence of inhibitors or the absence of promoters of bone resorption in mutant serum. In addition, the response of mutant and normal bone to parathyroid extract and hydrocortisone was tested in vitro. The results indicate that mutant and normal serum do not differ with respect to their ability to support bone resorption and that ia bone responds to hydrocortisone but not parathyroid extract in organ culture. These data indicate that the skeletal defect in ia rats is not humoral but cellular.